1. Field of the Invention
This invention pertains to protecting groups suitable for the protection of aminoacylated peptide moieties used to prepare misacylated tRNA's and to derivatize amino acids so as to facilitate the recovery of optically pure compounds. A family of compounds based on the 4-pentenoyl group, which forms a stable bound with the N.sup..alpha. site of amino acids has proved suitable for use both as a protecting group, and a derivatizing group for the optical separation of enantiomers. N-substituted 2-amino 4-pentenoyl derivatives are obtained, and can be used for the preparation of misacylated tRNA. Such suppressor tRNA can be used to elaborate proteins with synthetic amino acids at predetermined sites. In particular, N-substituted 2-amino-4-pentenoyl derivatives can be used for this process. The protecting group, if so used, is easily removed by treatment with iodine, with respect to which the protected amino acid and biologically effective groups in general are stable.
2. Background of the Prior Art
In recent years, the elaboration of proteins containing synthetic amino acids at predetermined sites has become technically feasible; the strategy employed involves readthrough of nonsense codons, Noren, et al., P.G. Science 244:182 (1989) with misacylated suppressor tRNA's. Hecht, et al., S.J. Biol. Chem. 253:4517 (1978). As first shown by Hecht and coworkers, Heckler, et al., S.M. J. Biol. Chem. 258:4492 (1983), Heckler, et al., S.M. Biochemistry 23:1468 (1984), misacylated tRNA's are accessible by T4 RNA ligase-mediated coupling of aminoacylated pCpA derivatives with tRNA's from which the 3'-terminal dinucleotide has been removed (FIG. 1).
While T4 RNA ligase-mediated ligation has been effected using unprotected aminoacyl-pCpA derivatives, Baldini, et al., J. Biochemistry 27:7959 (1988), the lability of the aminoacyl moiety has led most workers to employ amino acid protecting groups that impart chemical stability. Although numerous groups have been described in the literature as amine protecting groups, Greene, et al., Protecting Groups in Organic Synthesis 2.sup.nd Ed., John Wiley and Sons, New York, (1991), relatively few are suitable for the protecting of aminoacyl-pCpA derivatives. Almost none of the reported groups can be removed under conditions compatible with the integrity of the derived aminoacyl-tRNA's. The most satisfactory ones reported to date have been the pyroglutamyl Roesser, et al., S.M. Biochemistry 28:5185 (1989), and the nitroveratryloxycarbonyl (NVOC) Robertson, et al., J. Am. Chem. Soc. 113:2722 (1991) groups, removable by enzymatic proteolysis and photolytic cleavage, respectively. Baldini, et al. J. Biochemistry 27:7959 (1988). No protecting group removable with facility by a simple chemical treatment has been reported. Mendel, et al. J. Am. Chem. Soc. 113:2758 (1991).
Another problem, frequently encountered, in the synthesis of misacylated suppressor tRNA's is the chirality of the amino acids. Most interesting unnatural amino acids are obtained as racemic mixtures, or at best in an enantiomeric excess of one of the optical isomers. Some interesting methods for the asymmetric synthesis of amino acids have been developed, Williams, R. M., Synthesis of Optically Active .alpha.-Amino Acids Pergamon, Oxford, (1989), but few are stereospecific and most require additional separation in order to obtain optically pure derivatives.
Accordingly, it continues to be an object of those of skill in the art to provide and to address these problems we sought a group that would be suitable for the protection of N.sup..alpha. of aminoacyl-pCpA derivatives and at the same time would allow us to obtain optically pure derivatives.